Inducible nitric oxide synthase (iNOS) is a NOS isoform that is involved in the inflammatory response. iNOS may govern key mechanisms in the evolution of injury, protection and repair, including vascular regulation, inflammation, cytoprotection, cytotoxicity, and regeneration. In the traumatic brain injury (TBI), iNOS is expressed in a variety of cell types in the peri-trauma region. We performed studies examining long-term outcome after TBI and observed a powerful endogenous neuroprotectant effect of iNOS in two species. Our studies are supported by an expanding body of literature revealing important beneficial effects of iNOS in response to injury inside and outside of the CNS. The hypothesis of this proposal is that iNOS is expressed that TBI and is a powerful endogenous neuroprotectant. Specific aims are as follows: 1) Determine the time course, magnitude, and cellular localization of iNOS induction after experimental TBI in both mice and rats, 2) Test whether iNOS is an endogenous neuroprotectant and improves both histopathological and functional outcome after TBI, using both iNOS KO mice and iNOS inhibitors in rats. Also investigate the possibility that there is a biphasic role of iNOS after TBI, with early detrimental effects, but beneficial effects overall, 3) Test in both mice and rats if over-expression of iNOS by gene transfer with adenovirus-based vector is neuroprotective after TBI, 4) Determine in our mouse and rat models how iNOS confers its neuroprotective effects, including evaluation of downstream mediators such as cytokines, nerve growth factor (NGF), and cerebral blood flow (CBF), and 5) Define, in humans with severe TBI, the global and local production of NO, as assessed by nitrite/nitrate levels in cerebrospinal fluid (CSF) and brain interstitial fluid, respectively, and determine the time course, magnitude, and cellular localization of iNOS induction in human cerebral contusions. Our established CCI models will be used to produce TBI in mice and rats. Expression of iNOS will be studied using RT-PCR, enzyme activity, and immunohistochemistry. Two inhibitors of iNOS (aminoguanidine and N6-(ininoethyl1)-L-lysine) and iNOS KO mice will be used. A replication deficient adenovirus that expresses human iNOS will be used to transfect brain regions in vivo, both before and after injury. Outcome evaluation will include motor and cognitive (Morris water maze) tasks, histopathology, CBF (perfusion NMR), cytokines and NGF (ELISA), and macrophage/lymphocyte infiltration (by immunohistochemistry and flow cytometric analysis). In humans with severe TBI, nitrite/nitrate levels will be used as a marker of NO in both CSF and brain interstitial fluid (microdialysis). Brain samples from patients undergoing emergency resection of contusion, will be studied using immunohistochemistry. Confirming that iNOS is a neuroprotectant, showing that over-expression of iNOS as beneficial, and defining the mechanisms involved in this effect are key steps toward the of a novel treatment. Finally, these studies will unite bench to bedside for this important mechanism in TBI.